Nitric Oxide-Sensitive Guanylyl Cyclase Is Differentially Regulated by Nuclear and Non-Nuclear Estrogen Pathways in Anterior Pituitary Gland

17β-estradiol (E2) regulates hormonal release as well as proliferation and cell death in the pituitary. The main nitric oxide receptor, nitric oxide sensitive- or soluble guanylyl cyclase (sGC), is a heterodimer composed of two subunits, α and β, that catalyses cGMP formation. α1β1 is the most abundant and widely expressed heterodimer, showing the greater activity. Previously we have shown that E2 decreased sGC activity but exerts opposite effects on sGC subunits increasing α1 and decreasing β1 mRNA and protein levels. In the present work we investigate the mechanisms by which E2 differentially regulates sGC subunits' expression on rat anterior pituitary gland. Experiments were performed on primary cultures of anterior pituitary cells from adult female Wistar rats at random stages of estrous cycle. After 6 h of E2 treatment, α1 mRNA and protein expression is increased while β1 levels are down-regulated. E2 effects on sGC expression are partially dependent on de novo transcription while de novo translation is fully required. E2 treatment decreased HuR mRNA stabilization factor and increased AUF1 p37 mRNA destabilization factor. E2-elicited β1 mRNA decrease correlates with a mRNA destabilization environment in the anterior pituitary gland. On the other hand, after 6 h of treatment, E2-BSA (1 nM) and E2-dendrimer conjugate (EDC, 1 nM) were unable to modify α1 or β1 mRNA levels, showing that nuclear receptor is involved in E2 actions. However, at earlier times (3 h), 1 nM EDC causes a transient decrease of α1 in a PI3k-dependent fashion. Our results show for the first time that E2 is able to exert opposite actions in the anterior pituitary gland, depending on the activation of classical or non-classical pathways. Thus, E2 can also modify sGC expression through membrane-initiated signals bringing to light a new point of regulation in NO/sGC pathway

Photography-based taxonomy is inadequate, unnecessary, and potentially harmful for biological sciences

The question whether taxonomic descriptions naming new animal species without type specimen(s) deposited in collections should be accepted for publication by scientific journals and allowed by the Code has already been discussed in Zootaxa (Dubois & Nemésio 2007; Donegan 2008, 2009; Nemésio 2009a–b; Dubois 2009; Gentile & Snell 2009; Minelli 2009; Cianferoni & Bartolozzi 2016; Amorim et al. 2016). This question was again raised in a letter supported by 35 signatories published in the journal Nature (Pape et al. 2016) on 15 September 2016. On 25 September 2016, the following rebuttal (strictly limited to 300 words as per the editorial rules of Nature) was submitted to Nature, which on 18 October 2016 refused to publish it. As we think this problem is a very important one for zoological taxonomy, this text is published here exactly as submitted to Nature, followed by the list of the 493 taxonomists and collection-based researchers who signed it in the short time span from 20 September to 6 October 2016

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Nitric Oxide Plays a Key Role in Ovariectomy-Induced Apoptosis in Anterior Pituitary: Interplay between Nitric Oxide Pathway and Estrogen.

Changes in the estrogenic status produce deep changes in pituitary physiology, mainly because estrogens (E2) are one of the main regulators of pituitary cell population. Also, E2 negatively regulate pituitary neuronal nitric oxide synthase (nNOS) activity and expression and may thereby modulate the production of nitric oxide (NO), an important regulator of cell death and survival. Little is known about how ovary ablation affects anterior pituitary cell remodelling and molecular mechanisms that regulate this process have not yet been elucidated. In this work we used freshly dispersed anterior pituitaries as well as cell cultures from ovariectomized female rats in order to study whether E2 deficiency induces apoptosis in the anterior pituitary cells, the role of NO in this process and effects of E2 on the NO pathway. Our results showed that cell activity gradually decreases after ovariectomy (OVX) as a consequence of cell death, which is completely prevented by a pan-caspase inhibitor. Furthermore, there is an increase of fragmented nuclei and DNA cleavage thereby presenting the first direct evidence of the existence of apoptosis in the anterior pituitary gland after OVX. NO production and soluble guanylyl cyclase (sGC) expression in anterior pituitary cells increased concomitantly to the apoptosis. Inhibition of both, NO synthase (NOS) and sGC activities prevented the drop of cell viability after OVX, showing for the first time that increased NO levels and sGC activity observed post-OVX play a key role in the induction of apoptosis. Conversely, E2 and prolactin treatments decreased nNOS expression and activity in pituitary cells from OVX rats in a time- and E2 receptor-dependent manner, thus suggesting interplay between NO and E2 pathways in anterior pituitary

Cadmium induced-oxidative stress in pituitary gland is reversed by removing the contamination source

Cadmium (Cd2+) is one of the most important environmental contaminants and acts as an endocrine disruptor. Previously, we have demonstrated that the simultaneous administration of Cd2+ and melatonin (Mel) in drinking water impaired metal-induced oxidative stress in rat anterior pituitary gland. The aim of this study was to investigate if a treatment started after the toxic manifestations of Cd 2+ became evident could reverse the effects of the metal. Animals exposed to Cd2+ (5 parts per million [ppm], 30 days) were treated with Mel or without the metal during the next 1 or 2 months. Cd2+ exposure increased the expression of heme oxygenase-1 (HO-1), a biomarker of oxidative stress, and an a posteriori Mel treatment reversed oxidative stress induced by Cd2+. This effect was also observed 1 month after metal removal. The Cd2+-induced increase in metallothionein-1 (MT-1) and nitric oxide synthase 1 (NOS1) expression were also reversed by metal removal. In addition, serum prolactin and luteinizing hormone levels affected by Cd 2+ exposure were normalized. Considering that the manifestations of Cd2+ intoxication become evident only after a certain period of metal accumulation, these results show that metal removal is enough to reverse Cd2+ effects in anterior pituitary gland and bring to light the relevance of moving away the individual from the contamination source.Fil: Miler, Eliana Andrea. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina; ArgentinaFil: Nudler, Silvana Iris. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina; ArgentinaFil: Quinteros, Fernanda A.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina; ArgentinaFil: Cabilla, Jimena Paula. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina; ArgentinaFil: Ronchetti, Sonia Alejandra. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina; ArgentinaFil: Duvilanski, Beatriz Haydee. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina; Argentin

E2 down-regulates nNOS expression and activity in anterior pituitary cells through ER.

<p>Anterior pituitary cells from 14 days-OVX rats were stabilized for 24 h and incubated with or without 1 nM E2 for 24, 48 or 72 h. (<b>A</b>) nNOS mRNA levels were determined by RT-PCR. (<b>B</b>), NOx concentration was assayed by nitrate reductase-Griess assay. (<b>C</b>), Pituitary cell cultures from OVX rats were incubated with 1 nM E2 and/or with 100 nM ICI 182,780 (an estrogen receptor antagonist), 30 min before E2 treatment. nNOS mRNA levels were determined by RT-PCR. Results represent media ± SE of densitometric values relative to β-actin or nitrite concentration (N = 3). ANOVA followed by Tukey’s test, *p<0.05, **p<0.01 vs. respective control, Δp<0.05 vs. E2.</p

Prolactin (PRL) down-regulates nNOS expression and NO production in anterior pituitary cells.

<p><b>(A, B)</b> Anterior pituitary cells from 14 days-OVX rats were stabilized for 24 h and incubated with or without 500 ng/mL PRL for 24, 48 or 72 h. (<b>A</b>) nNOS mRNA levels were determined by RT-PCR. <b>(B)</b>, NOx concentration was assayed by nitrate reductase-Griess assay. (<b>C</b>) Pituitary cell cultures from 14 days-OVX rats were incubated for 48 h with 20 μM Tyrphostin AG490 (Tyr), a Jak2 protein kinase inhibitor, 30 min before 1 nM E2 or 500 ng/mL PRL treatments. nNOS protein levels were determined by western blot (N = 3). Results represent media ± SE of densitometric values relative to β-actin or nitrite concentration (N = 3). ANOVA followed by Tukey’s test, *p<0.05, **p<0.01 vs. respective control; Δp<0.05 vs. Tyr.</p

E2 and NO pathway cross-talk in anterior pituitary cells.

<p>In anterior pituitary cells, nNOS-mediated NO production is primarily associated with cell death by apoptosis. On the other hand, E2 is the main stimulator of PRL secretion, and promotes lactotroph survival and proliferation. NO and E2 pathways reciprocally inhibit each other at multiple points. NO through cGMP inhibits PRL secretion. E2 inhibits nNOS through hypothalamic GnRH inhibition and further down-regulates nNOS either directly or indirectly at pituitary level, through PRL stimulation. In addition, E2 inhibits sGC activity and promotes an imbalance in its subunits’ expression which has been associated with other functions, not related to cGMP production.</p

sGC expression is augmented after OVX and mediates NO-induced pituitary cell death.

<p>Anterior pituitary primary cell cultures from control and 14-days OVX rats were stabilized for 24 h. <b>(A)</b> α1 and β1 protein levels were measured by western blot after 48 h. <b>(B)</b>, α2 and α2i mRNA levels were determined by RT-PCR. (<b>C, D</b>) Cells were incubated with or without 10 μM LY-83583 (a sGC inhibitor) for 48 h. (<b>C</b>) Cell viability was determined by MTT assay. (<b>D</b>) Active caspase-3 protein levels were measured by western blot. Results represent mean ± SE, (N = 3) of relative units corresponding to the protein or mRNA densitometric values relative to β-actin expressed as percentage of control. ANOVA followed by Tukey’s test, *p<0.05, **p<0.01 vs. control, ΔΔp<0.01 vs. 14 days-OVX rats.</p